Capillary array and related methods

ABSTRACT

The invention provides methods and devices for detecting the presence of one or more target analytes in a sample employing a channel having affixed therein one or more binding partners for each target analyte. Assays are carried out by transporting the sample through the channel to each successive binding partner so that target analyte present in said sample binds to the corresponding binding partner. The sample is then transported beyond the binding partner(s), followed by detection of any target analyte bound to each binding partner. In one embodiment, binding efficiency is increased by the use of segmented transport, wherein a first bolus or bubble of a fluid that is immiscible with the sample precedes the sample during transport and a second bolus or bubble of a fluid that is immiscible with the sample follows the sample. Many configurations are possible for the device of the invention. A preferred device includes: a substrate with a channel formed in its surface, and a cover element that overlies and seals the channel. Binding partner(s) are affixed to the surface of the cover element facing the channel lumen.

CROSS-REFERENCES TO RELATED APPLICATION

This application is a divisional of and claims the benefit of U.S.application Ser. No. 10/418,384, filed Apr. 17, 2003 now U.S. Pat. No.6,818,184, which is a divisional of Ser. No. 09/652,873, filed Aug. 31,2000 now U.S. Pat. No. 6,610,499, the disclosures of which areincorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. CA58207,awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

This invention relates to the field of diagnostics. In particular thisinvention provides devices and methods that allow rapid detection and/orquantitation of multiple analytes.

BACKGROUND OF THE INVENTION

Tumors progress through the continuous accumulation of genetic andepigenetic changes that enable escape from normal cellular andenvironmental controls. These aberrations may involve genes that affectcell-cycle control, apoptosis, angiogenesis, adhesion, transmembranesignaling, DNA repair, and genomic stability. A number of genes thatcontribute to this process have already been discovered. However,large-scale analysis of gene expression and gene copy number suggestthat the number of such genes may be large, perhaps stingly so, and manyimportant cancer-related genes remain to be discovered. Identificationof recurrent changes in gene copy number, organization, sequence orexpression is one common approach to identification of genes that play arole in cancer. Large-scale array analysis techniques for assessment ofgenome copy number, expression level and DNA sequence polymorphisms arenow accelerating the rate at which tumors can be analyzed. These sametechnologies are promising as diagnostic platforms that can be employedto assess specific changes in individual tumors thereby permittingselection of therapeutic strategies that are optimal for these tumors.

Array based comparative genomic hybridization (CGH), allows the changesin relative DNA sequence copy number to be mapped onto arrays of clonedprobes. In array CGH, total genome DNAs from tumor and reference samplesare independently labeled with different fluorochromes or haptens andco-hybridized to normal chromosome preparations along with excessunlabeled Cot-1 DNA to inhibit hybridization of labeled repeatedsequences. The principle advantages of CGH are that it maps changes incopy number throughout a complex genome onto a normal reference genomeso the aberrations can be easily related to existing physical maps,genes and genomic DNA sequence. In addition, array CGH allowsquantitative assessment of DNA sequence dosage from one copy per testgenome to hundreds of copies per genome. Initial work involved CGH toarrays comprised of targets spanning >100 kb of genomic sequence, suchas BACs. More recently, CGH to cDNA arrays has been demonstrated. cDNAarrays are attractive for CGH since they are increasingly available andcarry a very large number of clones. In addition, the same array can beused to assess expression and copy number.

Single nucleotide polymorphisms (SNPs) also can be detected efficientlyby hybridization of fluorescently labeled PCR amplified representationsof the genome to arrays comprised of oligonucleotides. Both alleles ofeach of several thousand SNP markers and single-base mismatch targetsmay be presented on an array. The stringency of the hybridizationreaction is adjusted so that hybridization is diminished if a singlebase mismatch exists between the probe and oligonucleotide substrate.Thus, its hybridization signature can determine the presence or absenceof an allele in the hybridization mixture. This technique is rapid andscales well to genome-wide assessments of linkage or LOH (loss ofhomogeneity).

Enormous progress has been made in recent years in the development andDNA sequence characterization of cDNA clones from the human, mouse andother model organisms. In humans, these data have been computationallyassembled into over 8000 genes and 83,000 clusters. The cDNA clonesassociated with these sequences are publicly available. These clones andtheir associated sequences form the basis for a powerful microarrayapproach to large-scale analysis of gene expression. In this approach,labeled mRNA samples are hybridized to arrays of cDNA clones oroligonucleotides derived from the associated sequences. The arrays maybe on silicon or membrane substrates. The labeled probes may be labeledradioactively or with fluorescent reagents so that the resultinghybridization signals can be detected using autoradiography,phosphoimaging or fluorescence imaging.

cDNA and oligonucleotides arrays have been made using robots to move DNAfrom microtiter trays to silicon substrates or to nylon membranes. Thisapproach is flexible and is especially well-suited to production ofcustom arrays, but also has been applied to make large-scale arrayscarrying 40,000 different clones. An alternative is to synthesizeoligonucleotide arrays directly on silicon substrates usingphotolithographic approaches. These techniques work by projecting lightthrough a photolithographic mask onto the synthesis substrate. Singleoligonucleotide arrays on silicon substrates have been constructed withelements representing more than 40,000 genes/ESTs.

The conventional array approaches described above, while powerful, arelimited by the inefficient manner in which probe is used and by the longhybridization times required. These limitations arise from the need todistribute probe molecules over a large surface during hybridization. Asa result, most probe molecules are far from the targets to which theymight hybridize and sensitivity suffers. This reduces the rate at whichhybridization occurs and results in most probe molecules never reachingthe targets to which they might bind, a phenomenon that becomesincreasingly limiting for long oligonucleotides with slow diffusionrates. This problem can be reduced by using relatively large amounts ofprobe, vigorous mixing and using space-filling reagents such as dextransulfate in the hybridization mixtures. However, substantial improvementis still needed to allow practical use of DNA or RNA recovered fromsmall amounts of material (e.g., collected by microdissection) and toincrease the rate of hybridization.

SUMMARY OF THE INVENTION

The invention provides a method of detecting the presence of a firsttarget analyte in a sample. The method employs a channel having affixedtherein a first binding partner for the first target analyte. Thebinding partner is preferably an antibody, a binding protein, or anucleic acid. The method entails transporting the sample through thechannel to the first binding partner so that first target analytepresent in the sample binds to the first binding partner. A first bolusor bubble of a fluid that is immiscible with the sample precedes thesample during transport and a second bolus or bubble of a fluid that isimmiscible with the sample follows the sample during transport. Thesample is then transported beyond the first binding partner, and thepresence of any first target analyte bound to the first binding partneris detected.

In a preferred embodiment, the method employs a channel formed in asurface of a substrate. In a variation of this embodiment, a coverelement overlies and seals the channel and has a first surface facingthe channel lumen. Preferably, the cover element is removably attachedto the substrate. In a particularly preferred variation of thisembodiment, the channel has a hydrophobic lumenal surface. In this case,the first surface of the cover element is preferably hydrophilic. When acover element is present, the first binding partner is preferablyaffixed to the first surface of the cover element.

In a preferred embodiment, the immiscible fluids preceding and followingthe sample are gas bubbles. In a particularly preferred embodiment, afilm of fluid about 1 μm thick or less that contains the first targetanalyte forms between a gas bubble following the sample and a lumenalsurface of the channel or cover element, if present.

To enhance target analyte mixing and presentation to the bindingpartners affixed in the channel, the sample can be divided into at leasttwo segments that are separated by a bolus or bubble of a fluid that isimmiscible with the sample.

If desired, a buffer solution can follow the bolus or bubble ofimmiscible fluid that follows the sample. Like the sample, the buffersolution can be divided into at least two segments that are separated bya bolus or bubble of a fluid that is immiscible with the buffersolution.

The use of boluses or bubbles of immiscible fluid in the methods of theinvention improves the efficiency of target analyte-binding partnerbinding and therefore increases the speed at which assays can be run. Inpreferred embodiments, the sample is transported through the channel ata velocity of at least about 1 mm/second. Any fluid transport method canbe employed, but fluid is preferably transported by electrophoreticforce.

The invention also provides a device including a substrate; a channel ina surface of the substrate; a cover element that overlies and seals thechannel where the cover element has a first surface facing the channellumen; and a first binding partner for the first target analyte affixedto the first surface. The invention additionally provides a method ofdetecting the presence of a first target analyte in a sample thatemploys such a device. The method entails transporting the samplethrough the channel to the first binding partner so that first targetanalyte present in the sample binds to the first binding partner,transporting the sample beyond the first binding partner, and detectingthe presence of any first target analyte bound to the first bindingpartner.

Another device of the invention includes a channel defined by a channelwall, a member projecting into the channel lumen, and a first bindingpartner for the first target analyte affixed to the member. In preferredembodiments, the channel is a capillary tube, and the member is a fiberinserted into the capillary tube.

In preferred embodiments, devices of the invention include an electrodeto which a voltage can be applied to induce transport of the firsttarget analyte toward or away from the first binding partner.Preferably, a permeation layer overlies the electrode, and the firstbinding partner is attached to the permeation layer.

The devices and methods of the invention are particularly well-suitedfor conducting multi-analyte assays, in which case, the channel has aplurality of different binding partners affixed therein at distinctlocations.

In another aspect, the invention provides a method of producing an arrayof binding partners that entails introducing a bolus of a first bindingpartner into a channel, introducing a bolus or bubble of an immisciblefluid into the channel after the first binding partner, and introducinga bolus of a second binding partner into the channel after theimmiscible fluid. In a preferred embodiment, the channel is a loadingtube with a hydrophobic lumenal surface and each binding partner bolusis encapsulated in oil. This method additionally entails inserting theloading tube into an assay tube; transferring the first and secondbinding partners, separated by the bolus or bubble of immiscible fluid,into the assay tube; affixing the first and second binding partners to alumenal surface of the assay tube at distinct locations; and withdrawingthe loading tube from the assay tube.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method of attaching different binding partners todistinct locations in an assay tube according to the invention.

FIG. 2 illustrates a method of delivering different binding partners todistinct locations in an assay tube that minimizes “carryover” from onebinding partner to the next.

FIG. 3 is a schematic illustration of a device of invention in whichbinding partners are attached, via a permeation layer, to a centralelectrode that projects into the lumen of a channel.

FIG. 4 shows the mixing that occurs in a sample transported through achannel, when the sample is preceded and followed by a bolus or bubbleof an immiscible fluid.

FIG. 5 illustrates an embodiment of the invention in which thepresentation of target analyte to binding partners affixed to a channelis enhanced by the formation of a thin film of sample solutioncontaining the target analyte between a bolus or bubble of immisciblefluid following the sample.

FIG. 6 shows an embodiment of the invention in which a bolus of samplesolution is divided into segments by boluses or bubbles of immisciblefluid, as is a bolus of buffer solution following the sample solution.

FIG. 7 illustrates fluorescence detection of labeled target analytesbound to cognate binding partners in a capillary tube.

FIG. 8 is a schematic illustration of a device according to theinvention in which nucleic acid binding partners are affixed to a planarcover element, which is attached to a substrate including a channel.This device is described in Example 1.

FIG. 9 is an illustration of a modified version of the device of FIG. 8,which includes electrodes positioned under the nucleic acid bindingelements to provide electrophoretic enhancement of hybridization. Thisdevice is described in Example 2.

DETAILED DESCRIPTION I. Methods and Devices for Efficient Detection ofMultiple Analytes

This invention provides novel methods and devices for the rapiddetection and/or quantification of one or more target analytes in asample. In a preferred embodiment, the invention includes a channel inwhich a binding partner(s) is affixed. The binding partner(s) isspecific for an analyte to be detected Different binding partners canlocated at distinct locations in the channel so that binding of thecorresponding target analyte can be detected and/or quantified at eachbinding partner location.

II. Definitions

A “target analyte” is any molecule or molecules that are to be detectedand/or quantified in a sample. Preferred target analytes includebiomolecules such as nucleic acids, antibodies, proteins, sugars, andthe like.

The terms “binding partner” or “member of a binding pair” refer tomolecules that specifically bind other molecules to formula bindingcomplex such as antibody-antigen, lectin-carbohydrate, nucleicacid-nucleic acid, biotin-avidin, etc. In particularly preferredembodiments, the binding is predominantly mediated by non-covalent (e.g.ionic, hydrophobic, etc.) interactions. The terms “binding partner” and“member of a binding pair” apply to individual molecules, as well as toa set of multiple copies of such molecules, e.g., affixed to a distinctlocation of a surface. Thus, as used herein, the expression “differentbinding partners” includes sets of different binding partners, whereineach set includes multiple copies of one type of binding partner whichdiffers from the binding partners present in all other sets of bindingpartners.

The term “antibody,” as used herein, includes various forms of modifiedor altered antibodies, such as an intact immunoglobulin, an Fv fragmentcontaining only the light and heavy chain variable regions, an Fvfragment linked by a disulfide bond (Brinkmann et al. (1993) Proc. Natl.Acad. Sci. USA, 90: 547–551), an Fab or (Fab)′2 fragment containing thevariable regions and parts of the constant regions, a single-chainantibody and the like (Bird et al. (1988) Science 242: 424–426; Hustonet al. (1988) Proc. Nat. Acad. Sci. USA 85: 5879–5883). The antibody maybe of animal (especially mouse or rat) or human origin or may bechimeric (Morrison et al. (1984) Proc Nat. Acad. Sci. USA 81: 6851–6855)or humanized (Jones et al. (1986) Nature 321: 522–525, and published UKPatent Application No. 8707252).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to amino acid polymers containing onlynaturally occurring amino acids. The term “binding protein” refers toany protein binding partner other than an antibody, as defined above.

The terms “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Spri et al.(1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 141 9), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111:2321, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch,” Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994),Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp 169–176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

The term “specifically binds,” as used herein, when referring to atarget analyte (e.g., protein, nucleic acid, antibody, etc.), refers toa binding reaction that detects the presence of the target analyte in aheterogeneous population of molecules (e.g., proteins and otherbiologics). Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody or stringent hybridizationconditions in the case of a nucleic acid), the specified binding partnerbinds to its particular target analyte and does not bind in asignificant amount to other molecules present in the sample.

The terms “hybridizing specifically to” and “specific hybridization” and“selectively hybridize to,” as used herein refer to the binding,duplexing, or hybridizing of a nucleic acid molecule preferentially to aparticular nucleotide sequence under stringent conditions. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. Stringent hybridizationand stringent hybridization wash conditions in the context of nucleicacid hybridization are sequence dependent and are different underdifferent environmental parameters. An extensive guide to thehybridization of nucleic acids is found in, e.g., Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes part I, chapt 2, Overviewof principles of hybridization and the strategy of nucleic acid probeassays, Elsevier, N.Y. (“Tijssen”). Generally, highly stringenthybridization and wash conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Very stringent conditions areselected to be equal to the T_(m) for a particular probe. An example ofstringent hybridization conditions for hybridization of complementarynucleic acids which have more than 100 complementary residues on anarray or on a filter in a Southern or northern blot is 42° C. usingstandard hybridization solutions (see, e.g., Sambrook (1989) MolecularCloning: A Laboratory Manual (2nd ed.) Vol. 1–3, Cold Spring HarborLaboratory, Cold Spring Harbor Press, N.Y.), with the hybridizationbeing carried out overnight. An example of highly stringent washconditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example ofstringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes(see, e.g., Sambrook supra) for a description of SSC buffer). Often, ahigh stringency wash is preceded by a low stringency wash to removebackground probe signal. An example of a medium stringency wash for aduplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15minutes. An example of a low stringency wash for a duplex of, e.g., morethan 100 nucleotides, is 4× to 6×SSC at 40° C. for 15 minutes.

The term “channel” refers to a path that directs fluid flow in aparticular direction. The channel can be formed as a groove or trenchhaving a bottom and sides, or as a fully enclosed “tube.” In someembodiments, the channel need not even have “sides.” For example, ahydrophobic polymer can be applied to a flat surface and thereby confineand/or direct fluid flow on that surface in a narrow (e.g. hydrophilic)domain. The channel preferably includes at least one surface to which abinding partner can be affixed.

The term “microchannel” is used herein for a channel having acharacteristic dimension of about 100 μm or less.

The term “characteristic dimension” is used herein to denote thedimension that determines Reynolds number (Re), as is known in the art.For a cylindrical channel, it is the cross-sectional diameter. For arectangular channel, the characteristic dimension depends primarily onthe smaller of the width and depth. For a V-shaped channel it depends onthe width of the top of the “V,” and so forth. Calculation of Re, andthus characteristic dimensions, for channels of various morphologies canbe found in standard texts on fluid mechanics (e.g. Granger (1995) FluidMechanics, Dover, N.Y.; Meyer (1982) Introduction to Mathematical FluidDynamics, Dover, N.Y.).

The term “capillary tube” refers to a tube of small cross-sectionaldiameter. Open-ended capillary tubes of hydrophilic material, whencontacted with water, will typically take up the water by capillaryaction. Capillary tubes can be fabricated of a number of materialsincluding, but not limited to, glass, plastic, quartz, ceramic, andvarious silicates.

A “capillary electrophoresis tube” refers to a “capillary tube” designedfor and/or typically used or intended to be used in a capillaryelectrophoresis device.

The term “immiscible” refers to the absence of substantial mixingbetween two different fluids. Thus, a first fluid is immiscible in asecond when the two fluids are maintained separate fluid phases underthe conditions used.

As used herein with reference to binding partners, the term “distinctlocation” means that each binding partner is physically separated fromevery other binding partner such that a signal (e.g., a fluorescentsignal) from a labeled molecule bound to binding partner can be uniquelyattributed to binding at that binding partner.

As used herein, the term “electrophoretic force” is the force wherebyions in a fluid medium are transported toward an oppositely chargedelectrode in response to a voltage gradient.

The term “electroosmotic force” refers to that force whereby charges ina channel wall create a sheath of counterions in the adjacent fluid thatmoves the fluid column and solutes contained therein along the channelin response to a voltage gradient.

The term “array” refers to a collection of elements, wherein eachelement is uniquely identifiable. For example, the term can refer to asubstrate bearing an arrangement of elements, such that each element hasa physical location on the surface of the substrate that is distinctfrom the location of every other element. In such an array, each elementcan be identifiable simply by virtue of its location. Typical arrays ofthis type include elements arranged linearly or in a two-dimensionalmatrix.

III. Device Components

A. Channel

1. Channel Types and Dimensions

The device of to the invention includes a channel. Virtually any type ofchannel can be used in the invention. Appropriate channel types include,but are not limited to, tubes, grooves, channels formed by opposedbarriers, and the like. A preferred tube is a capillary tube, such as acapillary tube suitable for use in capillary electrophoresis. In apreferred device, the channel is a groove formed in the surface of asubstrate, and the device includes a cover element that overlies andseals the channel. In a variation of this embodiment, the cover elementis removably attached to the substrate. In an alternative device, thechannel is a capillary tube, and a member to which one or more bindingpartners is affixed projects into the channel lumen. In a variation ofthis embodiment, the member is a fiber inserted into the capillary tube.

The channel can have virtually any cross-section, e.g., circular,square, rectangular, triangular, V-shaped, U-shaped, hexagonal,octagonal, irregular, and so forth. The channel can have any convenientconfiguration including, but not limited to, linear, curved, serpentine(e.g., a linear portion joined by a curve or loop to another linearportion, which is itself joined by a curve or loop to a third linearbranch). In a preferred embodiment, the channel defines a serpentinepath, preferably one including linear portions aligned so that the longaxes of the linear portions are parallel. Such a channel is referred toherein as a “folded” channel. Folded channels of the invention caninclude as many linear portions as desired. The length of each linearportion can vary, depending on the application.

Any channel material is suitable for practice of this invention so longas the material is essentially stable to the solutions passed throughit. Preferred materials are capable of binding, or being derivatized tobind, the binding partner or a linker to the binding partner. Inaddition, in a preferred embodiment, the material is selected and/ormodified so that it does not substantially bind to the target analyte.Preferred materials also do not bind, or otherwise interact with, othercomponents (e.g. labels) whose binding would tend to increase the“background” signal in the assay methods of the invention. The sameconsiderations apply to a cover element or member that projects into thechannel lumen, if present. Glass or quartz cover elements areparticularly preferred for use in the devices of the invention.

In a preferred embodiment, the lumenal surface of the channel, or aportion thereof, is sufficiently hydrophobic to reduce the tendency ofan aqueous solution passing through the channel to leave behind aresidual film. In a particularly preferred embodiment, the channelincludes a hydrophilic lumenal surface, to which one or more bindingpartners is attached, and a hydrophobic lumenal surface, to which nobinding partners are attached. This embodiment is preferred when sampleis transported using segmented flow, as described in greater detailbelow. In a variation of this embodiment, the device has a channel witha hydrophobic lumenal surface and a cover element that overlies andseals the channel. A surface of the cover element facing the channellumen is preferably hydrophilic, and one or more binding partners areaffixed to this hydrophilic surface. Similarly, in a device including amember projecting into the channel lumen, a surface of the member ishydrophilic, and one or more binding partners are affixed thereto.

Particularly preferred channel/cover element/projecting member materialsinclude, but are not limited to, glass, silicon, quartz or otherminerals, plastic(s), ceramics, metals, paper, metalloids,semiconductive materials, cements, and the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. A wide variety oforganic and inorganic polymers, both natural and synthetic, can beemployed as channel materials. Illustrative polymers includepolyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF), polydimethylsiloxane(PDMS), silicones, polyformaldehyde, cellulose, cellulose acetate,nitrocellulose, and the like.

Polymeric channel materials can be rigid, semi-rigid, or non-rigid,opaque, semi-opaque, or transparent depending upon the use for whichthey are intended. For example, devices that include an optical orvisual detection element are generally fabricated, at least in part,from transparent materials to allow or at least facilitate thatdetection. Alternatively, transparent windows of, e.g., glass or quartzcan be incorporated into the device. Additionally, the polymericmaterials may have linear or branched backbones and may be crosslinkedor noncrosslinked. Example of particularly preferred polymeric materialsinclude, e.g., polydimethylsiloxane (PDMS), polyurethane,polyvinylchloride (VPC), polystyrene, polysulfone, polycarbonate, andthe like.

Conductive or semiconductive materials preferably include an insulatinglayer on the lumenal surface of the channel. This is particularlyimportant where the device incorporates electrical elements (e.g.,electrical fluid direction systems, electrical sensors, and the like).

If the device includes cover element sealing a channel, the coverelement and channel materials should be selected to provide asufficiently tight seal to prevent fluid loss during use. In onevariation of this embodiment, one or more binding partners are attachedto a surface of the cover element that faces the channel. Afterconducting an assay, any analyte(s) bound to the binding partner(s) canbe detected by removing the cover element and placing the cover elementin a detector. In this case, the cover element is preferably designed tofacilitate analyte detection. If, for example, the analyte is labeledwith a light absorbing label, such as, e.g., a fluorescent label, thecover element is preferably fabricated from a material that provides alow background signal in the detection system. Thus, where fluorescentlabels are used, a material having a low level of autofluorescence,e.g., glass, is employed in the cover element. Similar considerationsapply to the projecting member in devices wherein the binding partner(s)are affixed to a member, such as a fiber, projecting into the channellumen.

The dimensions of the channel are preferably as small as possible,consistent with ease of handling and mechanical stability, to reduce theamount of sample required for an assay and to reduce the distances thatanalyte must travel to reach a binding partner affixed in the channel.The preferred channel characteristic dimension range is between about0.5 μm and about 100 mm. Particularly preferred channels range from acharacteristic dimension of about 1 μm to about 5 mm. More preferably,the channel is a microchannel, e.g., with a characteristic dimensionbetween about 5 μm to about 100 μm. A most preferred characteristicdimension range is between about 5 μm and 50 μm. The channel length willdepend on the channel type, configuration, characteristic dimension, andlocation and number of binding partners. Preferred channels are lessthan about 500 cm, more preferably about 1 μm to about 300 cm, and evenmore preferably about 1 cm to about 100 cm.

The channel can be a component of a larger article. Thus, the channelcan be assembled with one or more other channels to provide amultiplicity of channels whereby a number of different assays can be runsimultaneously. The channel can also be a component of an instrumentthat includes appropriate liquid handling, and/or detection, and/orsample processing/application functions. If desired, channel(s)according to the invention can be fabricated as part of a reusable ordisposable unit that can be conveniently “plugged” into an instrumentfor running the assays of this invention.

It will be appreciated that the channel(s) can be provided on any of awide variety of articles including, but not limited to a microtiter dish(e.g., PVC, polypropylene, or polystyrene), a test tube (glass orplastic), a dipstick (e.g. glass, PVC, polypropylene, polystyrene,latex, and the like), a microcentrifuge tube, or a glass, silica,plastic, metallic or polymer bead. In particularly preferredembodiments, one or more channels are provided as a capillary channel ona glass or silicon slide, as a capillary tube (e.g. a capillaryelectrophoresis tube), or fabricated as an element of an “integratedcircuit” having on board circuit elements for control of sampleapplication, liquid flow, and/or signal detection.

2. Channel Fabrication

Methods of fabricating the channels of this invention are well known tothose of skill in the art. For example, where the channel is formed ofone or more capillary tubes, the capillaries can be purchased fromcommercial vendors (e.g. Polymicron Technologies, Tucson, Ariz.) orpulled or extruded by conventional capillary “pulling” machines.

Where the channels are fabricated on a surface, they can be formed usingstandard techniques, e.g., they can be machined, molded, carved, etched,laminated, extruded, or deposited, etc.

In a preferred embodiment, the channel(s) are fabricated usingmicromachining processes (e.g., photolithography) well known in thesolid-state electronics industry. Microdevices, e.g., microchannels, arecommonly constructed from semiconductor material substrates such ascrystalline silicon, widely available in the form of a semiconductorwafer used to produce integrated circuits, or from glass. Fabrication ofmicrodevices from a semiconductor wafer substrate can take advantage ofthe extensive experience in both surface and bulk etching techniquesdeveloped by the semiconductor processing industry for integratedcircuit (IC) production.

Surface etching, used in IC production for defining thin surfacepatterns in a semiconductor wafer, can be modified to allow forsacrificial undercut etching of thin layers of semiconductor materialsto create movable elements. Bulk etching, typically used in ICproduction when deep trenches are formed in a wafer using anisotropicetch processes, can be used to precisely machine edges or trenches inmicrodevices. Both surface and bulk etching of wafers can proceed with“wet processing,” using chemicals such as potassium hydroxide insolution to remove non-masked material from a wafer. For microdeviceconstruction, it is even possible to employ anisotropic wet processingtechniques that rely on differential crystallographic orientations ofmaterials, or to use electrochemical etch stops, to define variouschannel elements.

“Dry etch processing” is another technique that allows great flexibilityin microdevice design. This processing technique is particularlysuitable for anistropic etching of fine structures. Dry etch processingencompasses many gas or plasma phase etching techniques ranging fromhighly anisotropic sputtering processes that bombard a wafer with highenergy atoms or ions to displace wafer atoms into vapor phase (e.g., ionbeam milling), to somewhat isotropic low energy plasma techniques thatdirect a plasma stream containing chemically reactive ions against awafer to induce formation of volatile reaction products.

Intermediate between high energy sputtering techniques and low energyplasma techniques is a particularly useful dry etch process known asreactive ion etching. Reactive ion etching involves directing an ioncontaining plasma stream against a semiconductor, or other, wafer forsimultaneous sputtering and plasma etching. Reactive ion etching retainssome of the advantages of anisotropy associated with sputtering, whilestill providing reactive plasma ions for formation of vapor phasereaction products in response to contacting the reactive plasma ionswith the wafer. In practice, the rate of wafer material removal isgreatly enhanced relative to either sputtering techniques or low energyplasma techniques taken alone. Reactive ion etching therefore has thepotential to be a superior etching process for construction ofmicrodevices, with relatively high anistropic etching rates beingsustainable. The micromachining techniques described above, as well asmany others, are well known to those of skill in the art (see, e.g.,Choudhury (1997) The Handbook of Microlithography, Micromachining, andMicrofabrication, Soc. Photo-Optical Instru. Engineer, Bard & Faulkner(1997) Fundamentals of Microfabrication). In addition, examples of theuse of micromachining techniques on silicon or borosilicate glass chipscan be found in U.S. Pat. Nos. 5,194,133, 5,132,012, 4,908,112, and4,891,120.

In one embodiment, the channel is micromachined in a silicon (100) waferusing standard photolithography techniques to pattern the channels andconnection ports. Ethylene-diamine, pyrocatechol (EDP) is used for atwo-step etch and a Pyrex 7740 coverplate can be anodically bonded tothe face of the silicon to provide a closed liquid system. In thisinstance, liquid connections can be made on the backside of the silicon.

In other embodiments, the channel can be built up by depositing materialon a substrate to form channel walls (e.g., using sputtering or otherdeposition technology) or the channel can be cast/molded in a material.Cast/molded channels are easily fabricated from a wide variety ofmaterials including but not limited to various metals, plastics, orglasses. In certain preferred embodiments, the channel(s) are cast invarious elastomers. (e.g., alkylated chlorosulfonated polyethylene(Acsium®), polyolefin elastomers (e.g., Engage®), chlorosulfonatedpolyethylene (e.g., Hypalon®), perfluoroelastomer (e.g.: Kalrez®),neoprene-polychloroprene, ethylene-propylene-diene terpolymers (EPDM),chlorinated polyethylene (e.g., Tyrin®), various siloxane polymers (e.g.polydimethylsiloxane), etc.).

Microscopic channels can be produced in PDMS by a method that relies onoxidation of PDMS in oxygen plasma (See Anal. Chem. 70:4974(1998).)Oxidized PDMS seals irreversibly to other materials used in microfluidicsystems, such as glass, silicon oxide, and oxidized polystyrene.

B. Binding Partners

One or more binding partners that specifically bind a target analyte tobe detected are affixed in the channel(s) of the invention. The bindingpartner(s) used in this invention are selected based upon the targetanalytes that are to be identified/quantified. Thus, for example, wherethe target analyte is a nucleic acid the binding partner is preferably anucleic acid or a nucleic acid binding protein. Where the target analyteis a protein, the binding partner is preferably a receptor, a ligand, oran antibody that specifically binds that protein. Where the targetanalyte is a sugar or glycoprotein, the binding partner is preferably alectin, and so forth. A device of the invention can include severaldifferent types of binding partners, for example, multiple nucleic acidsof different sequence and/or nucleic acids combined with proteins in thesame device. The latter would facilitate, e.g., simultaneous monitoringof gene expression at the mRNA and protein levels. Other combinations ofdifferent types of binding partners can be envisioned by those of skillin the art and are within the scope of the invention. Methods ofsynthesizing or isolating such binding partners are well known to thoseof skill in the art.

1. Preparation of Binding Partners

a. Nucleic Acids

Nucleic acids for use as binding partners in this invention can beproduced or isolated according to any of a number of methods well knownto those of skill in the art. In one embodiment, the nucleic acid can bean isolated naturally occurring nucleic acid (e.g., genomic DNA, cDNA,mRNA, etc.). Methods of isolating naturally occurring nucleic acids arewell known to those of skill in the art (see, e.g., Sambrook et al.(1989) Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1–3, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Nucleic acids useful in the invention can also be amplified from anucleic acid sample. A number of amplification techniques have beendescribed, but the polymerase chain reaction (PCR) is the most widelyused. PCR is described in U.S. Pat. Nos. 4,683,202, 4,683,195,4,800,159, and 4,965,188, as well as in Saiki (1985) Science 230:1350.PCR entails hybridizing two primers to substantially complementarysequences that flank a target sequence in a nucleic acid. A repetitiveseries of reaction steps involving template denaturation, primerannealing, and extension of the annealed primers by a DNA polymeraseresults in the geometric accumulation of the target sequence, whosetermini are defined by the 5′ ends of the primers. As denaturation istypically carried out at temperatures that denature most DNA polymerases(e.g., about 93° C.–95° C.), a thermostable polymerase, such as thosederived from Thermus thermophilus, Thermus aquaticus (Taq), or Thermusflavus, is typically used for extension to avoid the need to addadditional polymerase for each extension cycle.

In a preferred embodiment, the nucleic acid is created de novo, e.g.,through chemical synthesis. In a preferred variation of this embodiment,nucleic acids (e.g., oligonucleotides) are chemically synthesizedaccording to the solid phase phosphoramidite triester method describedby Beaucage and Caruthers (1981) Tetrahedron Letts. 22(20): 1859–1862,e.g., using an automated synthesizer, as described inNeedham-VanDevanter et al. (1984) Nucleic Acids Res. 12: 6159–6168.Purification of oligonucleotides, where necessary, is typicallyperformed by either native acrylamide gel electrophoresis or byanion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom.255: 137–149. The sequence of the synthetic oligonucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Meth.Enzymol. 65: 499–560.

b. Antibodies/Antibody Fragments

Antibodies or antibody fragments for use as binding partners can beproduced by a number of methods well known to those of skill in the art(see, e.g., Harlow & Lane (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, and Asai (1993) Methods in Cell Biology Vol.37: Antibodies in Cell Biology, Academic Press, Inc. N.Y.). In oneembodiment, antibodies are produced by immunizing an animal (e.g. arabbit) with an immunogen containing the epitope to be detected. Anumber of immunogens may be used to produce specifically reactiveantibodies. Recombinant proteins are the preferred immunogens for theproduction of the corresponding monoclonal or polyclonal antibodies.Naturally occurring protein may also be used either in pure or impureform. Synthetic peptides are also suitable and can be made usingstandard peptide synthesis chemistry (see, e.g., Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3–284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149–2156, andStewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill.)

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen is mixed with an adjuvant andan animals is immunized. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the immunogen. When appropriately high titers ofantibody to the immunogen are obtained, blood is collected from theanimal and an antiserum is prepared. If desired, the antiserum can befurther fractionated to enrich for antibodies having the desiredreactivity. (See Harlow and Lane, supra).

Monoclonal antibodies can be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (See, Kohler and Milstein (1976) Eur. J. Immunol. 6:511–519). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yields of the monoclonal antibodiesproduced by such cells can be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, DNA sequences encoding a monoclonal antibody or a bindingfragment thereof can be isolated by screening a DNA library from human Bcells according to the general protocol outlined by Huse et al. (1989)Science, 246:1275–1281. Such sequences can then be expressedrecombinantly.

Antibodies fragments, e.g., single chain antibodies (scFv or others),can also be produced/selected using phage display technology. Theability to express antibody fragments on the surface of viruses thatinfect bacteria (bacteriophage or phage) makes it possible to isolate asingle binding antibody fragment from a library of greater than 10¹⁰nonbinding clones. To express antibody fragments on the surface of phage(phage display), an antibody fragment gene is inserted into the geneencoding a phage surface protein (pIII) and the antibody fragment-pIIIfusion protein is displayed on the phage surface (McCafferty et al.(1990) Nature, 348: 552–554; Hoogenboom et al. (1991) Nucleic Acids Res.19:4133–4137).

Since the antibody fragments on the surface of the phage are functional,phage bearing antigen binding antibody fragments can be separated fromnon-binding phage by antigen affinity chromatography (McCafferty et al.(1990) Nature, 348: 552–554). Depending on the affinity of the antibodyfragment, enrichment factors of 20 fold-1,000,000 fold are obtained fora single round of affinity selection. By infecting bacteria with theeluted phage, however, more phage can be grown and subjected to anotherround of selection. In this way, an enrichment of 1000 fold in one roundcan become 1,000,000 fold in two rounds of selection (McCafferty et al.(1990) Nature, 348: 552–554). Thus, even when enrichments are low (Markset al. (1991) J. Mol. Biol. 222: 581–597), multiple rounds of affinityselection can lead to the isolation of rare phage. Since selection ofthe phage antibody library on antigen results in enrichment, themajority of clones bind antigen after as few as three to four rounds ofselection. Thus, only a relatively small number of clones (severalhundred) need to be analyzed for binding to antigen.

Human antibodies can be produced without prior immunization bydisplaying very large and diverse V-gene repertoires on phage (Marks etal. (1991) J. Mol. Biol. 222: 581–597). In one embodiment, natural V_(H)and V_(L) repertoires present in human peripheral blood lymphocytes areisolated from unimmunized donors by PCR. The V-gene repertoires arespliced together at random using PCR to create a scFv gene repertoirewhich is then cloned into a phage vector to create a library of 30million phage antibodies (Marks et al. (1991) J. Mol. Biol. 222:581–597; Marks et al. (1993). Bio/Technology. 10: 779–783; Griffiths etal. (1993) EMBO J. 12: 725–734; Clackson et al. (1991) Nature. 352:624–628). It is also possible to isolate antibodies against cell surfaceantigens by selecting directly on intact cells. The antibody fragmentsare highly specific for the antigen used for selection and haveaffinities in the 1 μM to 100 nM range (Marks et al. (1991) J. Mol.Biol. 222: 581–597; Griffiths et al. (1993) EMBO J. 12: 725–734). Largerphage antibody libraries result in the isolation of more antibodies ofhigher binding affinity to a greater proportion of antigens.

c. Binding Proteins

In one embodiment, the binding partner can be a binding protein.Suitable binding proteins include, but are not limited to, receptors(e.g., cell surface receptors), receptor ligands (e.g., cytolines,growth factors, etc.), transcription factors and other nucleic acidbinding proteins, as well as members of binding pairs, such asbiotin-avidin.

Binding proteins useful in the invention can be isolated from naturalsources, mutagenized from isolated proteins, or synthesized de novo.Means of isolating naturally occurring proteins are well known to thoseof skill in the art. Such methods include, but are not limited to,conventional protein purification methods including ammonium sulfateprecipitation, affinity chromatography, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, (1982) ProteinPurification, Springer-Verlag, N.Y.; Deutscher (1990) Methods inEnzynology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y.). Where the protein binds a target reversibly, affinity columnsbearing the target can be used to affinity purify the protein.Alternatively the protein can be recombinantly expressed with a HIS-Tagand purified using Ni²+/NTA chromatography.

In another embodiment, the binding protein can be chemically synthesizedusing standard chemical peptide synthesis techniques. Where the desiredsubsequences are relatively short, the molecule may be synthesized as asingle contiguous polypeptide. Where larger molecules are desired,subsequences can be synthesized separately (in one or more units) andthen fused by condensation of the amino terminus of one molecule withthe carboxyl terminus of the other molecule thereby forming a peptidebond. This is typically accomplished using the same chemistry (e.g.,Fmoc, Tboc) used to couple single amino acids in commercial peptidesynthesizers.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield (1962)Solid-Phase Peptide Synthesis; pp. 3–284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149–2156, andStewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill.

In a preferred embodiment, the binding protein can also be producedusing recombinant DNA methodology. Generally this involves generating aDNA sequence that encodes the binding protein, placing the DNA sequencein an expression cassette under the control of a particular promoter,expressing the protein in a host, isolating the expressed protein and,if necessary, renaturing the protein.

DNA encoding binding proteins or subsequences of this invention can beprepared by any suitable method as described above, including, forexample, cloning and restriction of appropriate sequences or directchemical synthesis by methods such as the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68: 90–99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68: 109–151; thediethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,22: 1859–1862; and the solid support method of U.S. Pat. No. 4,458,066.

DNA encoding the desired binding protein(s) can be expressed in avariety of host cells, including E. coli, other bacterial hosts, yeast,and various higher eukaryotic cells, such as the COS, CHO and HeLa cellslines and myeloma cell lines. The DNA sequence encoding the bindingprotein is operably linked to appropriate expression control sequencesfor each host to produce an expression construct. For E. coli, examplesof appropriate expression control sequences include a promoter such asthe T7, trp, or lambda promoters, a ribosome binding site and preferablya transcription termination signal. For eukaryotic cells, such controlsequences can include a promoter, an enhancer derived, e.g., fromimmunoglobulin genes, SV40, cytomegalovirus, etc, and a polyadenylationsequence, and may include splice donor and acceptor sequences.

The expression vector can be transferred into the chosen host cell bywell known methods such as calcium chloride transformation for E. coliand calcium phosphate treatment or electroporation for mammalian cells.Cells transformed with the expression vector can be selected byresistance to antibiotics conferred by genes contained on the plasmids,such as the amp, gpt, neo and hyg genes.

Once expressed, the recombinant binding proteins can be purified usingconventional techniques, as described above.

d. Sugars and Carbohydrates

Other binding partners suitable for use in the invention include sugarsand carbohydrates. Sugars and carbohydrates can be isolated from naturalsources, enzymatically synthesized or chemically synthesized. Specificoligosaccharide structures can be produced using theglycosyltransferases that produce these structures in vivo. Such enzymescan be used as regio- and stereoselective catalysts for the in vitrosynthesis of oligosaccharides (Ichikawa et al. (1992) Anal. Biochem.202: 215–238). Sialyltransferase can be used in combination withadditional glycosyltransferases. For example, one can use a combinationof sialyltransferase and galactosyltransferases. A number of methods ofusing glycosyltransferases to synthesize desired oligosaccharidestructures are known Exemplary methods are described, for insance, WO96/32491, Ito et al. (1993) Pure Appl. Chem. 65:753, and U.S. Pat. Nos.5,352,670, 5,374,541, and 5,545,553. The enzymes and substrates can becombined in an initial reaction mixture, or alternatively, the enzymesand reagents for a second glycosyltransferase cycle can be added to thereaction mixture as the first glycosyltransferase cycle nearscompletion. By conducting two glycosyltransferase cycles in sequence ina single vessel, overall yields are improved over procedures in which anintermediate species is isolated.

Methods of chemical synthesis are described by Zhang et al. (1999) J.Am. Chem. Soc., 121(4): 734–753. Briefly, in this approach, a set ofsugar-based building blocks is created with each block preloaded withdifferent protecting groups. The building blocks are ranked byreactivity of each protecting group. A computer program then determinesexactly which building blocks must be added to the reaction so that thesequence of reactions from fastest to slowest produces the desiredcompound.

2. Attachment of Binding Partners

Binding partner(s) are affixed in the channel(s) of the invention so asto be capable of binding the corresponding target analyte(s). Thelinkage between the binding partner and the substrate is preferablychemically stable under assay conditions and hydrophilic enough to befreely soluble in aqueous solutions. In addition, the linkage shouldpreferably not produce significant non-specific binding of targetanalyte(s) to the substrate. Many methods for immobilizing molecules toa variety of substrates are known in the art. For example, the bindingpartner can be covalently bound or noncovalently attached throughspecific or nonspecific bonding.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups that may be present on thesubstrate surface and used for linking can include carboxylic acids,aldehydes, amino groups, cyano groups, ethylenic groups, hydroxylgroups, mercapto groups and the like. The manner of covalently linking awide variety of compounds to various surfaces is well known and is amplyillustrated in the literature. See, for example, Ichiro Chibata (1978)Immobilized Enzymes, Halsted Press, New York, and Cuatrecasas, (1970) J.Biol. Chem. 245:3059.

In addition to covalent bonding, various methods for noncovalentlybonding a binding partner can be used. Noncovalent binding is typically,but not necessarily, nonspecific absorption of a compound to thesurface. Typically, the surface is blocked with a second compound toprevent nonspecific binding of labeled assay components. Alternatively,the surface is designed such that it nonspecifically binds one componentbut does not significantly bind another. For example, a surface bearinga lectin such as concanavalin A will bind a carbohydrate containingcompound but not an unglycosylated protein. Various substrates for usein noncovalent attachment of assay components are reviewed in U.S. Pat.Nos. 4,447,576 and 4,254,082.

Where the binding partner is a nucleic acid or a polypeptide, themolecule can be chemically synthesized in situ, if desired. In situnucleic acid or protein synthesis typically involves standard chemicalsynthesis methods, substituting photo-labile protecting groups for theusual protecting groups (e.g., dimethoxy trityl group (DMT) used innucleic acid synthesis). Irradiation of the substrate surface atdiscrete locations results in selective coupling of the monomer (e.g.,nucleotide or amino acid) to the growing nucleic acid(s) orpolypeptide(s) at the irradiated site. Methods of light-directed polymersynthesis are well known to those of skill in the art (see, e.g., U.S.Pat. No. 5,143,854; PCT Publication Nos. WO 90/15070, WO 92/10092 and WO93/09668; and Fodor et al. (1991) Science, 251, 767–77).

In preferred embodiments, the binding partner is immobilized by the useof a linker (e.g. a homo- or heterobifunctional linker). Linkerssuitable for joining biological binding partners are well known. Forexample, a nucleic acid or protein molecule may be linked by any of avariety of linkers including, but not limited to a peptide linker, astraight or branched chain carbon chain linker, or by a heterocycliccarbon linker. Heterobifunctional cross linking reagents such as activeesters of N-ethylmaleimide have been widely used (see, for example,Lerner et al. (1981) Proc. Nat. Acad. Sci. USA, 78: 3403–3407 andKitagawa et al. (1976) J. Biochem., 79: 233–236, and Birch and Lennox(1995) Chapter 4 in Monoclonal Antibodies: Principles and Applications,Wiley-Liss, N.Y.).

In a preferred embodiment, the binding partner is immobilized utilizinga biotin/avidin interaction. In this embodiment, biotin or avidin with aphotolabile protecting group can be placed in the channel. Irradiationof the channel at a distinct location results in coupling of the biotinor avidin to the channel at that location. Then, a binding partnerbearing an avidin or biotin group, respectively, is contacted with thechannel, forms a biotin-avidin complex and is thus localized in theirradiated site. To affix multiple different binding partners todifferent locations, this process can be repeated at each bindingpartner location.

Another suitable photochemical binding approach is described by Sigristet al. (1992) Bio/Technology, 10: 1026–1028. In this approach, theinteraction of ligands with organic or inorganic surfaces is mediated byphotoactivatable polymers with carbene generatingtrifluoromethyl-aryl-diazirines that serve as linker molecules. Lightactivation of aryl-diazirino functions at 350 nm, yields highly reactivecarbenes, and covalent coupling is achieved by simultaneous carbeneinsertion into both the ligand and the inert surface. Thus, reactivefunctional groups are not required on either the ligand or supportingmaterial.

Binding partners can be affixed to any location within the channel thatcontacts the sample during an assay according to the invention. In apreferred embodiment, a device of the invention includes a cover elementthat overlies and seals the channel. In this case, the binding partnercan be attached to the surface of the cover element facing the channellumen. As discussed above, in preferred embodiments, the bindingpartners are affixed to a lumenal surface of the channel or coverelement that has a hydrophilic character. In an alternative embodiment,the binding partners are affixed to a member, such as a fiber, thatprojects into the channel lumen. Preferably, the binding partners areaffixed to a hydrophilic surface of the fiber.

Although devices of the invention need not include more than one type ofbinding partner, typically a plurality of different binding partners areaffixed in the channel (i.e., on the channel surface and/or on thesurface of the cover element or projecting member, if present) such thateach different type of binding partner occupies a distinct location.Illustrative devices of the invention contain between about 10 and about10⁶ different types of binding partners; e.g., devices having about 10²,about 10³, about 10⁴, and about 10⁵ binding partners can readily beproduced Such devices allow the simultaneous assay of multiple targetanalytes.

The dimensions of, and spacing between, binding parters should allowdetection of distinct signals from target analyte(s) bound to eachbinding partner. In an embodiment wherein binding partners are attachedto a lumenal channel surface, the channel has an internal diameter ofabout 1 mm to about 5 mm, preferably about 2 mm, and the bindingpartners occupy a region that has a length (along the channel axis) ofabout 100 μm to about 5 mm, preferably about 1 mm, although those ofskill in the art recognize that other lengths and center-to-centerdistances are possible. The center of each binding partner location isabout 1 mm to about 5 mm, preferably about 2 mm, from the center of eachadjacent binding partner location. In an alternative embodiment in whichbinding partners are attached to a cover element, the center-to-centerdistance between each binding partner location is preferably about 10 μmto about 5 mm, more preferably about 1 mm or less, even more preferablyabout 100 μm or less, and most preferably about 50 μm or less.

Where a removable cover element is employed, the binding partner(s) arepreferably affixed to the cover element. In preferred variations of thisembodiment, a plurality of different binding partners are affixed to thecover element at distinct locations facing the channel lumen to form anarray of binding partners, e.g., a linear array for a single, linearchannel or a two-dimensional array for a serpentine (folded) channel orfor a plurality of parallel channels.

Several methods are available for affixing binding partners to thelumenal surface of a channel. For example, binding partner solutions canbe aspirated into a channel separated by a bolus or bubble of a fluidthat is immiscible with the binding partner solutions. Mostconveniently, a first binding partner solution is aspirated into thechannel, followed by a quantity of air, followed by the next bindingpartner solution, and so forth, to produce a series of binding partnersseparated by air bubbles. As binding partners are transported along thechannel, some of each binding partner solution may be “carried over”from on binding partner to one or more following binding partners,leading to cross-contamination of binding partners. In this embodiment,the channel is preferably formed from a material that minimizes thisphenomenon. The amount of carryover can be estimated, e.g., according tothe method of Snyder and Adler (1976) Anal. Chem. 48:1022–1027. Briefly,the binding partner dispersion due to carryover in a tube can becalculated as follows:q=0.50πL _(t) d _(t) ²(uη/γ)^(2/3) V _(s)where q represents the retardation of the center of the distribution ofbinding partner in the segment of binding partner solution and alsoequals the variance (δ²) of the concentration distribution afterdispersion (ie., after flow is complete). Thus, q is a dimensionlessnumber representing the number of segments of displacement of the centerof the original (i.e., pre-flow) binding partner concentrationdistribution. The other variables in the equation are L_(t), which isthe length of the tube through which the binding partner segment istransported; dt, which is the lumenal diameter of the tube; u, which isthe velocity of segment transport through the tube, η, which is theviscosity of the binding partner solution; γ, which is the surfacetension between the solution and the tube; and V_(s), which is thevolume of the segment of binding partner solution. By estimatingcarryover, one skilled in the art can determine whethercross-contamination of binding partners is within acceptable limits forthe desired application. This will be the case for relatively shortchannels and/or those with a limited number of binding partners affixedtherein.

In one embodiment, after loading into the channel, binding partners areaffixed to a lumenal surface of the channel at distinct locations.Attachment of binding partners is conveniently achieved using photo- orheat-initiated chemistry. In this embodiment, binding partners and/orthe lumenal surface of the channel bear blocking moieties that can bephoto- or heat-activated to ink the binding partners to the lumenalsurface.

A strategy developed by Technicon Instruments Corporation can beemployed to provide on-line mixing of binding partners and cross linkingreagents (see, e.g., U.S. Pat. No. 4,853,336, issued Aug. 1, 1989 toSaros et al.). As applied to the present invention, and illustrated inFIG. 1, the binding partner 101 is aspirated into the loading tube 103,followed by a small bolus or bubble of immiscible fluid 102 and then abolus of cross linking reagent 104. The loading tube 103 is theninserted into a larger diameter assay tube 105 and the binding partner,immiscible fluid, and cross linking reagent are transferred to the assaytube. The choice of tube sizes will depend one the application. In apreferred embodiment, binding partners are loaded using a Teflon®loading tube with an internal diameter of 1.8 mm and a quartz assay tubewith an internal diameter of 1 mm. The bubble or bolus of immisciblefluid is too small to span the larger diameter of the assay tube, whichallows mixing between the binding partner and cross linking reagent. Ifmultiple reagents are required, they can be provided in one reagentbolus or separated by small boluses or bubbles of immiscible fluid (see,e.g. FIG. 1, showing second cross lining reagent 106).

An alternative embodiment that reduces carryover between one bindingpartner and the next is shown in FIG. 2. This technique employs aloading tube 203 with a hydrophobic (e.g., Teflon®) lumenal surface andis described, e.g. in U.S. Pat. No. 3,635,680 (issued Jan. 18, 1972 toPeoples et al.); U.S. Pat. No. 3,479,141 (issued Nov. 18, 1969 to Smytheet al.); U.S. Pat. No. 4,253,846 (issued Mar. 3, 1981 to Smythe et al.).Each binding partner bolus 201 is encapsulated in oil, which isconveniently accomplished by adding a small volume of low-density,hydrophobic oil, preferably a fluorocarbon oil, to the surface of thebinding partner solution before introduction into the loading tube. Theoil should be immiscible with the binding partner solution and shouldremain on the surface of the solution. Aspiration of a bolus of thebinding partner solution through the oil layer results in encapsulationof the binding partner bolus in oil. Withdrawal of the loading tube fromone oil-layered binding partner solution and transfer to the nextoil-layered binding partner solution typically results in the intake ofa volume of air. This process can be repeated, as desired, to produce aseries of binding partner segments 201 that are separated from the wallof the loading tube 203 by an oil layer and from one another by an airbubble 202.

Loading tube 203 is then inserted into an assay tube 204, e.g. a glassor quartz capillary tube, in which the binding partners will bedeposited. The binding partners, separated by boluses or bubbles ofimmiscible fluid, are transferred into the assay tube, typically viapositive or negative pressure (exerted, e.g., via a pump, such as aperistaltic pump). The oil generally clings to the hydrophobic loadingtube and/or moves to the end of the tube that is inserted into the assaytube, collecting there and, in some cases, traveling between the outersurface of the loading tube and the adjacent inner surface of the assaytube. If desired, one or more shallow grooves can be included in theouter surface of the loading tube to promote movement of the oil awayfrom the assay tube. During this transfer, the loading tube can be movedalong the assay tube, usually away from the last-deposited bindingpartner (ie., in the direction of withdrawal from the assay tube), todeposit the next binding partner with minimal carry over. Afterdeposition of all binding partners, the loading tube is withdrawn fromthe assay tube. In this embodiment, the lumenal surface of the assaytube can be functionalized such that binding partners bind to thesurface on contact. Alternatively, photo- or heat-activation can beemployed, as described above.

Excess unlinked binding partner can be removed by washing, and fixationcan be carried out, if desired.

Binding partners can be affixed to a cover element using standardtechniques for fabricating two-dimensional arrays, including, forexample, robotic spotting, inkjet printing, and photolithographictechniques. For example, U.S. Pat. No. 5,807,522 (issued Sep. 15, 1998to Brown and Shalon) describes a device that facilitates massfabrication of microarrays characterized by a large number ofmicro-sized assay regions separated by a distance of 50–200 microns orless and a well-defined amount of analyte (typically in the picomolarrange) associated with each region of the array. An alternative approachto robotic spotting uses an array of pins or capillary dispensers dippedinto the wells, e.g., the 96 wells of a microtiter plate, fortransferring an array of samples to a substrate.

Binding partners can be affixed to a projecting element, such as afiber, using any of a wide variety of suitable methods known to those ofskill in the art.

C. Electrode(s)

A device of the invention can optionally include one or more electrodes.In preferred embodiments, the electrode(s) are positioned in the devicesuch that a voltage applied to the electrode(s) can induceelectrophoretic and/or electroosmotic transport of a target analyterelative to a binding partner. Preferred devices employ electrophoretictransport. The use of electrodes to direct fluid transport inmicrofluidic devices is well known and described, for example, in U.S.Pat. No. 5,632,957 (issued May 27, 1997 to Heller et al.) (describingelectrophoretic transport) and U.S. Pat. No. 6,046,056 (issued Apr. 4,2000 to Parce et al.) (describing electroosmotic transport) and inFreemantle (1999) Science/Technology (1999) 77:27–36 and Gilles etal.(1999) Nature Biotechnology 17:365–370. In devices of the invention,electrodes can be placed at the termini of one or more channels. Theapplication of an electric field along the length of the channel inducescations to flow toward the negative electrode, and vice versa Moretypically, an electrode underlies one or more binding partners so that avoltage applied to the electrode causes target analyte to move towardand become concentrated in the vicinity of the binding partner tofacilitate binding. Unbound charged components present in the sample canbe induced to move away from the binding partner location by reversingthe charge polarity at the electrode. Thus, target analyte can be movedfrom one binding partner or group of binding partners to another bysequential modulation of voltages applied to multiple electrodes.

An electrode for directing fluid transport is conveniently positioned ona surface of the device facing the channel lumen. In embodimentsemploying a cover element, the electrode is preferably positioned on thecover element. As shown in FIG. 3, in embodiments employing a memberprojecting into the channel lumen, the projecting member can be made ofa metal or other conductive material, allowing the projecting member toserve as an electrode 304. The electrode 304 is coated with a permeationlayer 305, to which binding partners 301 are attached. In a variation ofthis embodiment, the electrode is encircled by semipermeable tube 306,which is itself inserted into the channel lumen. Second electrode 307 islocated in the annular space between inner, semipermeable tube 306 andchannel wall 308. In use, the inner tube contains the sample 303 and anynecessary buffer, wash, or reagent solutions, preferably separated byboluses or bubbles of immiscible fluid 302. The annular space betweenthe inner, semipermeable tube 306 and the channel wall 308 contains aconductive fluid. The semipermeable tube is not permeable to the targetanalytes, but allows passage of ions and smaller molecules.

The device usually includes a lead, for example, a platinum, chromium,or gold wire, connected to the electrode. In multi-electrodeembodiments, systems can be designed to modulate voltages at eachelectrode independently or at groups of electrodes simultaneously. Inaddition, systems can readily be designed to allow cycling of, and/orsequential modulation of, voltages at individual electrodes or groups ofelectrodes. Sequential modulation of voltages at electrodes positionedalong the length of the channel is particularly preferred to inducetarget analyte flow along the channel.

A permeation layer preferably overlies the electrode to separate thesample components from the harsh electrochemical environment near theelectrode. The permeation layer generally covers the entire surface ofthe electrode and has a thickness appropriate to the device, typicallyranging, e.g., from about 1 nm to about 500 μm, with about 500 nm toabout 50 μm being preferred. The permeation layer can be formed from anysuitable material, such as a polymer, ceramic, sol-gel, layeredcomposite material, clay, and controlled porosity glass. Preferably, thematerial used for the permeation layer has a porosity that excludestarget analyte molecules, but allows passage of ions and smallermolecules. In a preferred device designed for segmented flow, thepermeation layer is preferably hydrophilic.

The binding partner can be attached directly to the permeation layer orthe device can include an attachment layer overlying the permeationlayer. The attachment layer in this case is a material that is adaptedto provide a convenient surface for attaching the desired bindingpartner(s). Where the binding partner is a nucleic acid, the device can,for example, include an agarose permeation layer. If avidin is includedin this layer, a biotinylated nucleic acid binding partner can beattached to this layer. If the device contains multiple different typesof binding partners, an electrode can be positioned under each differentbinding partner location or an electrode can underlie several differentbinding partner locations.

In an alternative embodiment, electrochemical methods can be combinedwith redox-active surfactants, as described by Gallardo et al. (1999)283:57, to actively control the motions and positions of aqueous andorganic liquids on millimeter and smaller scales. Surfactant speciesgenerated at one electrode and consumed at another can be used tomanipulate the magnitude and direction of spatial gradients in surfacetension and guide liquids through fluidic networks.

IV. Integrated Assay Device

State-of-the-art chemical analysis systems for use in chemicalproduction, environmental analysis, medical diagnostics and basiclaboratory analysis are preferably capable of complete automation. Suchtotal analysis systems (TAS) (Fillipini et al. (1991) J. Biotechnol. 18:153; Garn et al (1989) Biotechnol. Bioeng. 34: 423; Tshulena (1988)Phys. Scr. T23: 293; Edmonds (1985) Trends Anal. Chem. 4: 220; Stinshoffet al. (1985) Anal. Chem. 57:114R; Guibault (1983) Anal. Chem Symp. Ser.17: 637; Widmer (1983) Trends Anal. Chem. 2: 8) automatically performfunctions ranging from introduction of sample into the system, transportof the sample through the system, sample preparation, separation,purification and detection, including data acquisition and evaluation.

Recently, sample preparation technologies have been successfully reducedto miniaturized formats. Thus, for example, gas chromatography (Widmeret al. (1984) Int. J. Environ. Anal. Chem. 18: 1), high pressure liquidchromatography (Muller et al. (1991) J. High Resolut. Chromatogr. 14:174; Manz et al. (1990) Sensors & Actuators B1:249; Novotny et al., eds.(1985) Microcolumn Separations: Columns, Instrumentation and AncillaryTechniques J. Chromatogr. Library, Vol. 30; Kucera, ed. (1984)Micro-Column High Performance Liquid Chromatography, Elsevier,Amsterdam; Scott, ed. (1984) Small Bore Liquid Chromatography Columns:Their Properties and Uses, Wiley, N.Y.; Jorgenson et al. (1983) J.Chromatogr. 255: 335; Knox et al. (1979) J. Chromatogr. 186:405; Tsudaet al. (1978) Anal. Chem. 50: 632) and capillary electrophoresis (Manzet al. (1992) J. Chromatogr. 593: 253; Olefirowicz et al. (1990) Anal.Chem. 62: 1872; Second Int'l Symp. High-Perf. Capillary Electrophoresis(1990) J. Chromatogr. 516; Ghowsi et al. (1990) Anal. Chem. 62:2714)have been reduced to miniaturized formats.

Similarly, in another embodiment, this invention provides an integratedassay device (e.g., a TAS) for detecting and/or quantifying one or moretarget analytes. The assay device comprises the channel(s) with attachedbinding partner(s) as described above. In addition, preferred integratedassay devices also include one or more of the following: sampleapplication well(s) and/or injection port(s), one or more reservoirs toprovide buffers and/or wash fluids, one or more electrodes that directfluid transport, a detector, a heating or cooling element to controlassay temperature, a computer controller (e.g., for control of sampleapplication, reservoir flow switching, fluid transport, signaldetection, and the like).

In a particularly preferred embodiment, the integrated assay devicecontains the channel(s) in a “removable” module that can be easilyinserted and removed from the ancillary equipment. Where the channelused in the device is a tube (e.g. a capillary electrophoresis tube), aconventional capillary electrophoresis device contains much of theancillary plumbing, sample handling and delivery components, andcomputer controller(s) for an “integrated” assay device according to thepresent invention.

V. Running Assays

In general, assays are run by introducing the sample into the channelhaving one or more affixed binding partners. The sample is transportedthrough the channel to the first binding partner. The sample contactsthe first binding partner under conditions that allow the bindingpartner to specifically bind any corresponding target analytes that maybe present in the sample. The sample is then transported beyond thefirst binding partner and the presence of target analyte bound to thefirst binding partner is detected.

In a preferred embodiment, the channel includes a second binding partneraffixed at a location distinct from that of the first binding partner.After contact with the first binding partner, the sample is transportedthrough the channel to the second binding partner. The second bindingpartner is usually, but need not be, a different type of binding partnerfrom the first binding partner, e.g., the binding partners can be twonucleic acids of different sequence. The sample contacts the secondbinding partner under conditions that allow specific binding of thesecond target analyte, after which the sample is transported beyond thesecond binding partner. The presence of second target analyte bound tothe second binding partner is detected, and preferably binding to thefirst and second binding partners is detected in a single step. Inpreferred embodiments, the assay employs a channel having a plurality ofdifferent binding partners affixed therein, as described above, whichfacilitates multi-analyte assays.

A. Sample Preparation

Virtually any sample can be analyzed using the devices and methods ofthis advantage. However, in a preferred embodiment, the sample is abiological sample. The term “biological sample,” as used herein, refersto a sample obtained from an organism or from components (e.g., cells)of an organism. The sample may be of any biological tissue or fluid.Frequently the sample will be a “clinical sample” which is a samplederived from a patient. Such samples include, but are not limited to,sputum, cerebrospinal fluid, blood, blood fractions (e.g. serum,plasma), blood cells (e.g., white cells), tissue or fine needle biopsysamples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.Biological samples may also include sections of tissues such as frozensections taken for histological purposes.

Biological samples, (e.g., serum) may be analyzed directly or they maybe subject to some preparation prior to use in the assays of thisinvention. Such preparation can include, but is not limited to,suspension/dilution of the sample in water or an appropriate buffer orremoval of cellular debris, e.g. by centrifugation, or selection ofparticular fractions of the sample before analysis. Nucleic acidsamples, for example, are typically isolated prior to assay and, in someembodiments, subjected to procedures, such as reverse transcriptionand/or amplification (e.g., polymerase chain reaction, PCR) to increasethe concentration of all sample nucleic acids (e.g., using randomprimers) or of specific types of nucleic acids (e.g., usingpolynucleotide-thymidylate to amplify messenger RNA or gene-specificprimers to amplify specific gene sequences).

Preferably, the biological samples are processed so that the targetanalyte(s) of interest are labeled with a detectable label. Detectablelabels suitable for use in the present invention include any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include biotin for staining with labeled streptavidinconjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g.,fluorescein, Texas red, rhodamine, green fluorescent protein, and thelike, see, e.g., Molecular Probes, Eugene, Oreg., USA), radiolabels(e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradishperoxidase, alkaline phosphatase and others commonly used in ELISAs),and calorimetric labels such as colloidal gold (e.g., gold particles inthe 40–80 nm diameter size range scatter green light with highefficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Suitable chromogens that can be employed in the invention include thosethat absorb light in a distinctive range of wavelengths so that a colorcan be observed or, alternatively, that emit light when irradiated withradiation of a particular wavelength or wavelength range, e.g.,fluorescent molecules. Preferably, the label is a light absorbing label.Fluorescent labels are particularly preferred because they provide verystrong signals with low background. Fluorescent labels are alsooptically detectable at high resolution and sensitivity through a quickscanning procedure. Fluorescent labels offer the additional advantagethat irradiation of a fluorescent label with light can produce aplurality of emissions. Thus, a single label can provide for a pluralityof measurable events.

Desirably, fluorescent labels should absorb light above about 300 nm,preferably above about 350 nm, and more preferably above about 400 nm,usually emitting at wavelengths greater than about 10 nm higher than thewavelength of the light absorbed. It should be noted that the absorptionand emission characteristics of the bound dye can differ from theunbound dye. Therefore, when referring to the various wavelength rangesand characteristics of the dyes, it is intended to indicate the dyes asemployed and not the dye that is unconjugated and characterized in anarbitrary solvent.

It will be recognized that fluorescent labels are not to be limited tosingle species organic molecules, but include inorganic molecules,multi-molecular mixtures of organic and/or inorganic molecules,crystals, heteropolymers, and the like. Thus, for example, CdSe-CdScore-shell nanocrystals enclosed in a silica shell can be easilyderivatized for coupling to a biological molecule (Bruchez et al. (1998)Science, 281: 2013–2016). Similarly, highly fluorescent quantum dots(zinc sulfide-capped cadmium selenide) have been covalently coupled tobiomolecules for use in ultrasensitive biological detection (Warren andNie (1998) Science, 281: 2016–2018).

Detectable signal can also be provided by chemiluminescent andbioluminescent labels. Chemiluminescent sources include a compound whichbecomes electronically excited by a chemical reaction and can then emitlight which serves as the detectable signal or donates energy to afluorescent acceptor. Alternatively, luciferins can be used inconjunction with luciferase or lucigenins to provide bioluminescence.

Spin labels are provided by reporter molecules with an unpaired electronspin which can be detected by electron spin resonance (ESR)spectroscopy. Exemplary spin labels include organic free radicals,transitional metal complexes, particularly vanadium, copper, iron, andmanganese, and the like. Exemplary spin labels include nitroxide freeradicals.

The target analyte(s) can be labeled before, during, or after the samplecontacts the channel-affixed binding partner. So-called “direct labels”are detectable labels that are directly attached to or incorporated intothe target analyte prior to binding with the cognate binding partner.“Indirect labels” are attached to a component capable of binding to thetarget analyte or to a member of a binding pair, the other member ofwhich is attached to the target analyte. In indirect labeling, thelabeled component can be linked to the target analyte before, during orafter the target analyte-containting sample contacts the bindingpartner. Thus, for example, the target analyte can be biotinylated andthen bound to the cognate binding partner. After binding, anavidin-conjugated fluorophore can bind the biotin-bearing targetanalyte, providing a label that is easily detected. This embodiment isparticularly preferred for labeling nucleic acids. Nucleic acids can bedirectly labeled via an in vitro transcription or an amplificationreaction. Thus, for example, fluorescein-labeled UTP and CTP can beincorporated into the RNA produced in an in vitro transcription. For adetailed review of methods of labeling nucleic acids and detectinglabeled hybridized nucleic acids see Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 24: Hybridization With NucleicAcid Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).

The labels can be attached to the target analyte directly or through alinker moiety. In general, the site of label or linker-label attachmentis not limited to any specific position. For example, in nucleic acidlabeling, a label may be attached to a nucleoside, nucleotide, oranalogue thereof at any position that does not interfere with detectionor hybridization as desired. For example, certain Label-On Reagents fromClontech (Palo Alto, Calif.) provide for labeling interspersedthroughout the phosphate backbone of an oligonucleotide and for terminallabeling at the 3′ and 5′ ends. Labels can be attached at positions onthe ribose ring or the ribose can be modified and even eliminated asdesired. The base moieties of useful labeling reagents can include thosethat are naturally occurring or modified in a manner that does notinterfere with their intended use. Modified bases include but are notlimited to 7-deaza A and G, 7-deaza-8-aza A and G, and otherheterocyclic moieties.

B. Sample Delivery

The sample can be introduced into the devices of the invention accordingto standard methods well known to those of skill in the art. Thus, forexample, the sample can be introduced into the channel through aninjection port such as those used in high pressure liquid chromatographysystems. In another embodiment the sample can be applied to a samplewell that communicates with the channel. In still another embodiment thesample can be pumped into the channel. Means of introducing samples intochannels are well known and standard in the capillary electrophoresisand chromatography arts.

C. Fluid Transport

Samples and/or carrier/buffer/wash fluids can be introduced into and/ormoved through the channel according to standard methods. For example,fluid can be introduced and moved through the channel by a simplegravity feed from a “reservoir.” Alternatively, fluids can be movedthrough the channel by gas pressure or by fluid pressure produced by anyof a variety of suitable pumps (e.g., peristaltic pumps, metering pumps,etc.), pressure on a deformable chamber/diaphragm, etc. Fluid can alsobe driven through the channel by electrophoretic and/or electroosmoticmethods, which are well known and described, for example in U.S. Pat.No. 5,632,957 (supra) and U.S. Pat. No. 6,046,056 (supra).

Fluid transport can be continuous or discontinuous. If continuoustransport is employed, the fluid velocity is typically set to ensurethat the sample remains in contact with each binding partner for a timesufficient for any cognate target analyte to bind. If desired, the assaycan employ discontinuous flow, where the sample is moved into contactwith a binding partner and maintained in this position for a timesufficient for binding. In one embodiment, the sample is moved intocontact with a binding partner and then moved slightly forward andslightly backward (or vice versa) to enhance mixing and bring moretarget analyte into contact with the binding partner. The “back andforth” movement can be repeated as desired to further enhance mixing.

Electrophoretic and/or electroosmotic methods typically employelectrodes that can be charged positive, negative, or neutral to inducemovement and/or concentration of target analytes in the vicinity of oneor more binding partners and/or bulk fluid flow through the channel. Ina preferred embodiment, the channel includes a plurality of electrodesarranged at distinct binding partner locations along the length of thechannel, and a voltage is applied to each electrode in sequence. Forexample, if the target analytes are nucleic acids, which are negativelycharged, a positive DC potential can be applied to each successiveelectrode, to induce the target analytes to move toward and concentratenear each successive binding partner. The positive potential can bemaintained for a time sufficient to enable an appropriate target analyteto bind the binding partner. The polarity at the electrode can then bereversed, to repel unbound target analyte away from the binding partnerand on to the next target element.

In a preferred embodiment, the charge polarity at the electrode isreversed several times to mix the target analyte near the electrode,which enhances the diffusion of target analyte to the binding partner,increasing the rate of accumulation of target analyte specifically boundto the binding partner. Charge polarity reversal can optionally becarried out at increasing potential to increase the stringency of theassay (electronic stringency). The process can be carried out until adesired stringency is reached. Stringency can be monitored, for example,by including an internal control in the assay system, i.e., the assaycan be run using one or more positive and/or negative control analytesfor the binding partner(s) affixed in the channel. Parameters such asthe magnitude and duration of the voltage pulse, as well as the numberand timing of pulses can be varied to achieve the desired stringency.Gilles et al., (1999) Nature Biotechnology 17:365–370, describe the useof electronic stringency in nucleic acid hybridization assays to removesignal attributable to “mismatch” control probes to background levels.

The rate of fluid transport will depend on the configuration of thedevice and the kinetics of the binding interaction being assayed. Asmore rapid assays are generally preferred, device and assay designparameters are usually selected to allow relatively rapid fluidtransport. Higher fluid velocities can be employed, if desired, usingsegmented transport to enhance mixing and to accelerate diffusion oftarget analytes to binding partners.

D. Segmented Transport

The use of segmented fluid transport in fluidic assay devices is wellknown and is described, for example, in U.S. Pat. No. 4,853,336 (issuedAug. 1, 1989 to Saros et al.). Successive liquid segments areestablished in a conduit that are separated from one another by animmiscible fluid. This technique has been used in systems that permitthe delayed on-line mixing of different components of an analysismixture, such as samples with reagents or diluents. Prior to the presentinvention, segmented fluid transport was used to promote the mixing andinteraction of fluid components in a conduit. In the methods of thisinvention, by contract, this technique is used to enhance the convectivemixing of target analyte in a sample moving through a channel and thepresentation of target analyte to substrate-affixed binding partners.

FIG. 4 shows an embodiment in which the sample 403 is transportedthrough the channel 401 to a first binding partner with a bolus orbubble of a fluid 402 that is immiscible with the sample preceding thesample during transport. Another bolus or bubble of immiscible fluid 402follows the sample during transport. The immiscible fluid(s) can be thesame or different and a preferably sufficiently immiscible that thesample is substantially maintained as a separate phase during transport.The immiscible fluids can be any fluid that does not contain componentsreactive with the channel or channel components that the immisciblefluids contact during the assay or with the target analytes, bindingpartners, or any other assay reagents (e.g., labels). Examples ofimmiscible fluids suitable for use in the invention include silicon oiland immiscible, non-reactive gases. Gases are preferred, with airbubbles being most conveniently employed.

If the assay requires exposure of the binding elements to more than justthe sample solution, e.g., buffers, wash solutions, labeling reagentsolutions, etc., each solution can be separated from any other solutionby a bolus or bubble of immiscible fluid.

In preferred embodiments, the sample is aqueous solution, and thelumenal surface of the channel is hydrophobic, except for a portion(s)of the channel to which one or more binding partners are affixed. In aparticularly preferred embodiment, the assay employs the device of theinvention having a cover element with a hydrophilic lumenal surface andchannel with a hydrophobic lumenal surface. During transport, there islittle carryover of the aqueous sample solution between immisciblefluid-isolated liquid segments. However, a small quantity of samplesolution adheres to the hydrophilic binding partner-bearing surface(s).The unbound portion of this carryover sample solution is “picked up” bya following liquid segment, e.g., one containing buffer, and presentedto subsequent binding partners. An important advantage of segmented flowis that the segment contents are stirred, as shown in FIG. 4, increasingthe amount of target analyte presented to each binding partner.

A preferred embodiment exploits the existence of a thin surface film ofsample solution between boluses or bubbles of immiscible fluid and thehydrophilic surface of the channel/cover element to enhance targetanalyte binding. In the field of Continuous Flow Analysis for whichsegmented flow was first developed (Skeggs (1957) American Journal ofClinical Pathology 28:311–322), the presence of the surface film betweenthe bubble and the channel wall was considered a cause of undesirablecarryover from one liquid segment to the following liquid segmentHowever, the present invention can take advantage of this carryover toprovide “thin-film” presentation of target analytes to binding partners.As liquid segments and boluses or bubbles move past, the surface film iscaught between the bolus/bubble and the hydrophilic surface and thenmoves from one liquid segment into the next liquid segment. Thethickness of the film formed between the bolus/bubble and thehydrophilic surface is defined by the following equation:d _(f)=0.5πd _(t)(uη/γ)^(2/3)where d_(f)=thickness of film; d_(t)=diameter of tube; u=velocity offlow; η=viscosity; γ=surface tension. See Snyder and Adler (1976) Anal.Chem. 48:1018–22; Snyder and Adler (1976) Anal. Chem. 48:1023–27.

FIG. 5 illustrates the situation in which the film captured betweenbolus or bubble 502 and the hydrophilic surface of channel 504 is caughtup by and mixes with following liquid segment 503. (Binding partners 501are also shown.) As is apparent from the above equation, the thicknessof the surface film can be manipulated by the surface tension, theviscosity of the sample solution and by the velocity of flow. With thecontrol available, the sample solution can be presented to bindingpartner(s) in a film sufficiently thin, on the order of 1 μm that targetanalyte diffusion to the hydrophilic surface is rapid.

FIG. 6 illustrates the use of multiple boluses or bubbles of immisciblefluid 602 to increase the proportion of the sample 603 presented as athin film and to increase the exchange of bulk solution in the samplebolus with the thin film between the bolus/bubble and the lumenalsurface. The sample solution 603 is divided into a number of segmentsseparated by small boluses/bubbles 602. As the segmented flow moves downthe channel, target analyte in the film adhering to the hydrophilicsurface that has not hybridized to a binding partner, is carried fromthe leading sample segment into the following segment, mixing with it.This transfer and mixing reoccurs between each adjacent sample segment,efficiently mixing the segments and exposing the target elements tofresh sample solution in a thin film. The transfer of sample solutionfrom one liquid segment to the next results in a slow dilution of thetarget analyte as the column of sample solution segments moves down thetube. However, buffer solution 601 following sample 603 can captureunbound target analyte and re-present it to the binding partners.Preferably, buffer solution 601 is divided into a number segmentsseparated by a bolus or bubble of immiscible fluid 602, as shown in FIG.6.

Thin-film presentation of target analyte makes it possible to run theassay at relatively high flow velocities. One of skill in the art canreadily determine suitable flow velocity by calculating the amount oftime required for a target analyte to diffuse to and bind its cognatebinding partner. For example, the flow velocity for a nucleic acidhybridization assay can be calculated as follows. If the target analyteis a 15-bp oligonucleotide, it has a length of 15*0.34 nm/bp, a diameterof 2.6 nm and a volume of 15*0.34 nm*(1.3 nm)²*π=27.1 nm³. A sphere ofequal volume has a radius of R=(3/(4π)*V)^(1/3)=1.86 nm, and the lateraldiffusion coefficient of a sphere (D) is δγηπ.D=kT/(6πηR)(η water, 20° C.)=0.01 Poise (poise=g/cm s)D=1.38*10⁻¹⁶ erg/K*293 K/(6*π*0.01 g/(cm s)*1.86*10⁻⁷cm=1.15*10⁻⁶ cm²/sAnd the distance diffused is:d=(2Dt)^(1/2)andt=d ²/2DThus for a 15-mer to diffuse 1μ:t=0.005 sec.Consequently, once a 1 μm thick film of sample solution has formedbetween a bubble and a surface, a 15-mer will require only 5 msec todiffuse to the surface and be within hybridization reach of targetsequences.

Given a diffusion time of 5 msec, a 1-mm long sample could move at avelocity of 1 mm/0.005 sec or 200 mm/sec. Allowing 100 diffusion timesfor hybridization to occur gives a flow velocity of 2 mm/sec. Thus,segmented flow through a channel will efficiently present targetanalytes to the binding partners in a 10,000-binding partner channel inabout 1–2 hours. Of course, where assay time is not a concern, assays ofthe invention can be run at lower flow velocities, e.g., about 10mm/sec, about 1 mm/sec, about 0.1 mm/sec, about 0.01 mm/sec, and about0.001 mm/sec.

E. Segmented Electrophoretic Transport

In some embodiments, fluid transport is combined with electrophoretictransport. Use of segmentation allows precise control over thepositioning of the sample or sample segment relative to the bindingpartner(s) and the corresponding electrode(s). Fluid transport can thusbe coordinated so that an electrode serving a binding partner locationis appropriately charged (ie., positively or negatively, depending onthe target analyte charge) when the sample or sample segment ispositioned over that binding element. By setting the size of theimmiscible fluid bolus or bubble so that it spans several electrodes,the electric field is confined to the target segment of interestpreventing electric current and electrolysis from occurring where it isnot useful. In this embodiment, one or more sensors (e.g., opticalsensors) can be employed to follow the progress of the sample and toswitch on the electrodes when appropriate.

F. Other Methods of Enhancing Target Analyte Mixing

In addition to the method described above, mixing and presentation oftarget analyte, or other assay components, to binding partners can beenhanced by including particles in the relevant fluids. A preferredembodiment includes particles in the sample and/or in a buffer solutiontransported through the channel after the sample. The particles shouldbe non-reactive with the assay and device components that the particleswill contact in use. The particles can be formed of any of the materialsdescribed above with respect to channel materials, as well as othermaterials known to those of skill in the art. Polymeric particles arepreferred and are available in a variety of shapes and sizes. Theparticles should be sized to enhance mixing. In embodiments employingsegmented flow, the particles must not be so large as to disrupt theboluses or bubbles of immiscible fluid. In a preferred embodiment, thechannel has a half-circular shape, an internal diameter of about 100 μm,and particles that are about 5 μm to about 20 μm, preferably about 10 μmto about 15 μm, are included in the sample solution to enhance mixing.

Mixing can also be enhanced by appropriate channel design. For example,the channel can include one or more irregularities or obstacles to flow,such as e.g. bumps, that induce turbulence. In devices designed forsegmented flow, the size and shape of such irregularities or obstaclesshould allow boluses or bubbles of immiscible fluid to pass by intact.In a half-circular channel having an internal diameter of about 100 μm,for example, the channel can include one or more bumps extending about15 μm to about 25 μm, preferably about 20 μm from the channel wall. Ifdesired, such irregularities can be spaced a set distance before bindingpartner locations to provide mixing specifically where it is need.

G. Binding Conditions

Once in the channel, the sample is held under conditions that promotespecific binding between the sample and the binding partner. Conditionscompatible with specific binding between a binding partner and a giventarget analyte are well known to those of skill in the art. For example,buffers suitable for promoting binding between an antibody and a targetprotein are well known in the immunoassay art (see, e.g. U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168; Asai (1993) Methods inCell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc.New York; Stites & Terr (1991) Basic and Clinical Immunology 7thEdition).

Similarly conditions under which nucleic acids specifically hybridize toeach other are also well known to those of skill in the art. (see, e.g.,Tijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 24: Hybridization With Nucleic Acid Probes, Elsevier,N.Y.).

Nucleic acid hybridization simply involves contacting single-strandednucleic acids under conditions where complementary nucleic acids canform stable hybrid duplexes through complementary base pairing. Thenucleic acids that do not form hybrid duplexes are then washed awayleaving the hybridized nucleic acids to be detected, typically throughdetection of an attached detectable label. It is generally recognizedthat nucleic acids are denatured by increasing the temperature ordecreasing the salt concentration of the buffer containing the nucleicacids, adding chemical agents, or the raising the pH. Under lowstringency conditions (e.g., low temperature and/or high salt) hybridduplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where theannealed sequences are not perfectly complementary. Thus specificity ofhybridization is reduced at lower stringency. Conversely, at higherstringency (e.g., higher temperature or lower salt) successfulhybridization requires fewer mismatches.

One of skill in the art will appreciate that hybridization conditionsmay be selected to provide any degree of stringency. In a preferredembodiment, hybridization is performed at low stringency to ensurehybridization and then subsequent washes are performed at higherstringency to eliminate mismatched hybrid duplexes. Successive washesmay be performed at increasingly higher stringency (e.g., down to as lowas 0.25×SSPE at 37° C. to 70° C.) until a desired level of hybridizationspecificity is obtained. Stringency can also be increased by addition ofagents such as formamide. Finally, electronic stringency can be employedas described above to achieve the desired stringency. Hybridizationspecificity can be evaluated by comparing hybridization of targetanalyte nucleic acids with hybridization of control nucleic acids thatcan be included in the hybridization mixture.

In general, there is a tradeoff between hybridization specificity(stringency) and signal intensity. Thus, in a preferred embodiment, thewash is performed at the highest stringency that produces consistentresults and that provides a signal intensity greater than approximately10% of the background intensity. This stringency can be determinedempirically by washing the hybridized target analyte nucleic acids withsuccessively higher stringency solutions and detecting binding aftereach wash. Analysis of the data sets thus produced will reveal a washstringency above which the hybridization pattern is not appreciablyaltered and which provides adequate signal for the assay.

In a preferred embodiment, background signal is reduced by the use of ablocking reagent (e.g., tRNA, sperm DNA, Cot-1 DNA, etc.) during thehybridization to reduce non-specific binding. The use of blocking agentsin hybridization is well known to those of skill in the art (see, e.g.,Chapter 8 in P. Tijssen, supra).

Optimal hybridization conditions are also a function of the sensitivityof label (e.g., fluorescence) detection for different combinations ofsubstrate type, fluorochrome, excitation and emission bands, spot sizeand the like. Low fluorescence background surfaces can be used (see,e.g., Chu (1992) Electrophoresis 13:105–114). The sensitivity fordetection of binding partner spots of various diameters on the candidatesurfaces can be readily determined by, e.g., spotting a dilution seriesof fluorescently end labeled DNA fragments. These spots are then imagedusing conventional fluorescence microscopy. The sensitivity, linearity,and dynamic range achievable from the various combinations offluorochrome and solid surfaces (e.g., glass, fused silica, etc.) canthus be determined. Serial dilutions of pairs of fluorochrome in knownrelative proportions can also be analyzed. This determines the accuracywith which fluorescence ratio measurements reflect actual fluorochromeratios over the dynamic range permitted by the detectors andfluorescence of the substrate upon which the binding partner has beenfixed.

H. Detection

Virtually any method of analyte detection can be used in accordance withthe methods of this invention. Methods of detecting target analytes arewell known to those of skill in the art. Where the target analyte islabeled, the analyte is detected by detecting the label. Alternatively,binding of the target analyte can be detected by detecting a physicalproperty of the target analyte. Preferably, the detection methodemployed is one that allows quantification of target analyte binding.

Since the identity of target analytes can determined by the location(s)the binding partner(s) to which they bind, there is no need to usedifferent labels to identify different analytes. Target analytes can bedirectly or indirectly labeled. Indirect labeling most typically entailsthe use of labeled component capable of binding to the target analyte,e.g., a labeled antibody. The labeled component can be a member of abinding pair, the other member of which is attached to the targetanalyte, e.g., biotin-avidin. Where indirect labeling is employed,binding between the labeled component and the target analyte can occurbefore, during or after binding of the target analyte to the bindingpartner affixed in the channel. The label can be detectable throughoutthe assay procedure or can be detectable as a result of interaction witha detection system that is delivered in a bolus of fluid that followsthe sample and any buffer or wash solutions.

Methods of detecting target analytes are well known to those of skill inthe art. Where the target analyte is labeled (e.g., with a radioactive,fluorescent, magnetic, or mass label), the analyte is detected bydetecting the label. Preferably, the target analyte(s) present in thesample are labeled with a light-absorbing label, such as a fluorescentlabel.

Fluorescent labels are conveniently detected using a standard readerincluding an excitation light source and a fluorescence detector. Aconventional reader can include, for example, a mercury arc lamp and aCCD camera to collect fluorescence intensity data (see, e.g., Pinkel etal. (1998) Nature Genetics 20:207–211). Multiple filters are typicallyemployed to collect intensity data for different fluorophores. Where thechannel is a tube, such as a capillary tube, fluorescent detection canbe carried out by passing the channel 701 through a reader, as shown inFIG. 7, where the light source 702 provides excitation light, and theresulting fluorescence signal is detected by a detector 703. Suitablesystems are available for analyzing fluorescence signals ontwo-dimensional microarrays, and these can be used to detect andquantify binding to a two-dimensional array of binding partners spottedon a cover element of the invention.

Depending on the detection method, a detector or component of adetection system can be incorporated into a device of the invention.Alternatively, the surface of the device bearing the targetanalyte-bound binding partners can be removed from the channel tofacilitate detection. For example, if the binding parmers are affixed tothe cover element, the cover element can be unsealed from the channeland signal detected using any detection system suitable for detectingbinding to microarrays, e.g., DNA microarrays.

Although assays of the invention can be used to detect multiple targetanalytes without the need for multiple labels, for some applications,multiple labels may be desirable. For example, the assays of theinvention can be used for comparative assays in which the sampleincludes target analytes derived from two or more different sources. Thetarget analytes from each source are labeled with a different label. Thedifferent labels should be readily distinguishable. For instance, targetanalyte derived from one source could have a green fluorescent label,and target analyte derived from another source could have a redfluorescent label. The detection step distinguishes sites binding thered label from those binding the green label. In this manner the bindingof differently labeled target analytes to a single binding partner canbe analyzed independently from one another.

The differently labeled target analytes can be mixed to form a samplethat is introduced into the channel as described above. Alternatively,the target analytes from one source can be introduced into the channelas a first sample, followed by introduction of the target analytes fromany other sources as separate samples. After binding to channel-affixedbinding partners, the signals from the labeled target analytes boundeach binding partner are detected. The intensities of any signalproduced by each different label at each binding partner location arecompared as an indication of the relative amounts of each type of targetanalyte in the original sources.

I. Comparative Genomic Hybridization/Expression Monitoring

In a preferred embodiment, a device according to the invention is usedin a Comparative Genomic Hybridization (CGH) or expression monitoringassay. CGH is a approach used to detect the presence and identify thechromosomal location of amplified or deleted nucleotide sequences. (See,Kallioniemi et al., Science 258: 818–821 (1992); WO 93/18186.) In thetraditional implementation of CGH, genomic DNA is isolated from normalreference cells, as well as from test cells (e.g., tumor cells). The twonucleic acids (DNA) are labeled with different labels and thenhybridized in situ to metaphase chromosomes of a reference cell. Therepetitive sequences in both the reference and test DNAs can be removedor their hybridization capacity can be reduced by some means such as anunlabeled blocking nucleic acid (e.g. Cot-1). Chromosomal regions in thetest cells that are at increased or decreased copy number can be quicklyidentified by detecting regions where the ratio of signal from the twoDNAs is altered. For example, those regions that have been decreased incopy number in the test cells will show relatively lower signal from thetest DNA than the reference compared to other regions of the genome.Regions that have been increased in copy number in the test cells willshow relatively higher signal from the test DNA.

In one embodiment, the present invention provides a CGH-type assay inwhich the device of the present invention replaces the metaphasechromosome used for hybridization target in traditional CGH. Instead,the binding partners affixed in the channel are nucleic acid sequencesselected from different regions of the genome. The device itself becomesa sort of “glass chromosome” where hybridization of a nucleic acid to aparticular binding partner is informationally equivalent tohybridization of that nucleic acid to the region on a metaphasechromosome from which the binding partner is derived. In addition,nucleic acid binding partners not normally contained in the genome, forexample viral nucleic acids, can be employed.

More particularly, in a CGH-type assay, a device of the invention can beutilized in methods for quantitatively comparing copy numbers of atleast two nucleic acid sequences in a first collection of nucleic acidsrelative to the copy numbers of those same sequences in a secondcollection. The binding partners for these nucleic acids can be any typeof nucleic acid, e.g., genomic DNA, cDNA, amplified DNA, synthetic DNA,or RNA particularly mRNA), as can the collections of nucleic acids. Inpreferred embodiments, the nucleic acid collections are genomic DNA, orrepresentations thereof (e.g. amplified sequences), and the copy numbercomparison yields information about copy number variations (ie.,amplifications and/or deletions) between the two nucleic acidcollections. In other preferred embodiments, the nucleic acidcollections are mRNA, or representations thereof (e.g., cDNA oramplified sequences), and the copy number comparison yields informationabout differences in levels of expression of particular genes betweenthe two nucleic acid collections. Similar types of comparative assayscan be performed by binding collections of proteins from differentsources to channel-affixed antibodies or other binding proteins. Suchembodiments are useful in expression monitoring studies.

If repetitive sequences are present in the hybridization mixture formedwhen the nucleic acid collection(s) contact channel-affixed nucleic acidbinding partners, unlabeled blocking nucleic acids (e.g., Cot-1 DNA) canbe included in the hybridization mixture. The blocking of repetitivesequence hybridization allows detection of so-called “unique sequence”copy number variation. Blocking nucleic acids can be mixed with thenucleic acid collections before introduction into the channel.Alternatively, a solution of blocking nucleic acids can precede a bolusor bubble of immiscible fluid preceding the sample.

In a typical embodiment, one collection of nucleic acids is preparedfrom a test cell, cell population, or tissue under study; and the secondcollection of nucleic acids is prepared from a reference cell, cellpopulation, or tissue. Reference cells can be normal non-diseased cells,or they can be from a sample of diseased tissue that serves as astandard for other aspects of the disease. For example, if the referencenucleic acids are genomic DNA isolated from normal cells, then the copynumber of each sequence in that genomic DNA relative to the others isknown (e.g., two copies of each autosomal sequence, and one or twocopies of each sex chromosomal sequence depending on gender). Comparisonof this to test nucleic acids permits detection of variations fromnormal. Alternatively the reference nucleic acids can be prepared fromgenomic DNA from a primary tumor that may exhibit substantial copynumber variations, and the test nucleic acids can be prepared fromgenomic DNA of metastatic cells from that tumor, so that the comparisonshows the differences between the primary tumor and its metastasis.Further, both collections of nucleic acids can be prepared from normalcells. For example comparison of mRNA populations between normal cellsof different tissues permits detection of differential gene expressionthat is a critical feature of tissue differentiation. Thus, the terms“test” and “reference” are used for convenience to distinguish the twocollections of nucleic acids; neither term is intended to imply anythingabout the characteristics of the nucleic acids.

VI. Kits for Multiple Analyte Detection

In one embodiment, the invention provides kits for screening for,identifying the presence or absence, and/or quantifying-one or moreanalytes in a sample. A kit of the invention includes a channel of theinvention including one or more binding partners affixed therein asdescribed above. The channel is preferably designed for simple and rapidincorporation into an integrated assay device, e.g., a device includingone or more of the following: sample application well(s) and/orinjection port(s), one or more reservoirs to provide buffers and/or washfluids, one or more electrodes that direct fluid transport, a detector,a computer controller. The kit can additionally include appropriatebuffers and other solutions and standards for use in the assay methodsdescribed herein.

In addition, a kit can include instructional materials containingdirections (i.e., protocols) for the practice of the methods of thisinvention. While the instructional materials are typically written orprinted materials, they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto, electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

All publications cited herein are incorporated by reference in theirentirety.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Capillary-Based Array Hybridization System

A capillary channel-based array hybridization system is illustrated inFIG. 8. The channel substrate 802 is composed of molded PDMS(polydimethylsiloxane) material, which can be molded with precise detailand which provides a surface readily sealed against the planar coverelement (coverplate) 803. The channel 804 has a half-circular crosssection. To minimize the amount of target analyte required for analysisand to minimize the distances through which target analytes must diffuseto reach binding partners, the channel has an internal diameter of 100μm and a total length of 1 m. The channel is folded 100 times, thedistance between the center of one channel segment and the center of anadjacent channel sequence is 400 μm, and the channel substrate is 4cm×10 cm.

The coverplate is glass. Nucleic acid binding partners 801 are printedon the coverplate with using a robotic arrayer robot conventionally usedfor production of standard 2-dimensional DNA microarrays. The center-tocenter distance between adjacent binding partners in the same row isapproximately 100 μm (rows run along the longitudinal axis of eachchannel segment when the coverplate is sealed over the channel. Thecenter-to-center distance between adjacent binding partners in the samecolumn is approximately 400 μm (columns are perpendicular to the rows).This approach has the advantage that existing facilities can implementthe capillary-array analysis strategy using existing array equipment.The coverplate can be easily removed for fluorescence analysis, and theflow channel can be reused multiple times.

A capillary-array CGH hybridization assay is carried out as describedpreviously for array CGH. Briefly, genomic DNA from a test source islabeled by nick translation with fluorescein dCTP, and reference genomicDNA is labeled by nick translation with Texas red dCTP. The labeled DNAsare mixed with excess unlabeled Cot-1 DNA and precipitated with ethanol.The precipitated DNA is resuspended in 50% formamide, 10% dextransulfate, 2×SSC, 2% sodium dodecyl sulfate (SDS) and 100 mg tRNA. The DNAis denatured at 70° C. and transported through the capillary channel at37° C. After hydridization, the coverplate is removed and washed in 50%formamide, 2×SSC, pH7, at 45° C. and once in 0.1 M soldium phosphatebuffer with 0.1% NP40, pH 8, at room temperature. Green/red fluorescenceratios are measured using an ACAS 570, confocal, scanning-laser system.

Example 2 Capillary-Based Array Electrophoretic Hybridization System

Referring to FIG. 9, a device having the basic configuration describedin Example 1 has nucleic acid binding partners 901 printed in a2-dimensional array on a glass coverplate 902 on top oftitanium-platinum electrodes 903. The electrodes 903 underlie a columnof different binding partner locations in the 2-dimensional array. Eachcolumn of different binding partner locations has a long axis that isgenerally perpendicular to the longitudinal axis of each straightsegment of the channel 904. To protect the nucleic acids fromelectrolysis products formed at the electrodes, the coverplate surface,including the conducting strips, are coated with a permeation layercomprised of 2% glyoxal agarose and 1 mg/ml streptavidin mixture. Thenucleic acid binding partners are synthesized with a biotin attached toone end. Once the binding partners are deposited on the permeationlayer, avidin/biotin binding affixes the nucleic acids, forming bindingpartner elements.

In use, a positive DC potential is applied to successive electrodestrips along the channel to attract negatively charged target nucleicacids to each successive binding partner along the capillary, presentingtarget nucleic acids to each nucleic acid binding partner forhybridization. The polarity is then reversed to repel unhybridizedmaterial, which then moves to the next binding partner in response thepositive DC potential at its associated electrode. Preferably, thepotential is reversed several times at each electrode to “mix” the probeand to define an electric “stringency” of hybridization.

1. A method of detecting the presence of a first target analyte in asample, said method comprising: (a) providing: a substrate; a channel ina surface of said substrate; a cover element that overlies and sealssaid channel, said cover element having a first surface facing thechannel; and a first binding partner for said first target analyteaffixed to said first surface; (b) transporting said sample through saidchannel to said first binding partner so that first target analytepresent in said sample binds to said first binding partner; (c)transporting said sample beyond said first binding partner; and (d)detecting the presence of any first target analyte bound to said firstbinding partner, wherein said cover element is unsealed from saidchannel to allow said detection.
 2. The method of claim 1, wherein saidchannel is molded in a polymeric material.
 3. The method of claim 2,wherein said polymeric material is a polydimethylsiloxane.
 4. The methodof claim 1, wherein said channel has a hydrophobic lumenal surface. 5.The method of claim 1, wherein said channel has a cross-sectionaldiameter of about 100 μm or less.
 6. The method of claim 1, wherein saidcover element is removably attached to said substrate.
 7. The method ofclaim 1, wherein said first surface of said cover element ishydrophilic.
 8. The method of claim 1, additionally comprising detectingthe presence of a second target analyte in a sample, wherein a secondbinding partner for a second target analyte is affixed to said firstsurface of said cover element at a location distinct from that of saidfirst binding partner; said sample is additionally transported throughsaid channel to said second binding partner under conditions whereinsecond target analyte present in said sample binds to said secondbinding partner; said sample is transported beyond said first and secondbinding partners; and the presence of any second target analyte bound tosaid second binding partner is detected.
 9. The method of claim 8,wherein at least about 100 different binding partners are affixed todistinct locations of said first surface of said cover element.
 10. Themethod of claim 1, wherein said sample is transported through saidchannel at a velocity of at least about 1 mm/second.
 11. The method ofclaim 1, wherein fluid is transported in response to a pressuredifference.
 12. The method of claim 1, wherein said fluid is transportedby electrophoretic force.
 13. The method of claim 1, wherein saidbinding partner is selected from the group consisting of an antibody, abinding protein, and a nucleic acid.
 14. The method of claim 1, whereinsaid first target analyte is labeled.
 15. The method of claim 1, whereinsaid first target analyte is labeled with a light-absorbing label. 16.The method of claim 1, wherein: target analytes derived from at leasttwo different sources are present in said sample or are transportedthrough said channel in a plurality of separate samples, wherein targetanalytes from each source are labeled with a different label; aplurality of different binding partners are affixed to distinctlocations of said first surface of said cover element; said detectingcomprises detecting the signals from labeled target analytes bound toeach different binding partner; and said method additionally comprisescomparing the intensities of any signal produced by each different labelat each different binding partner.